Coronaviruses are a well characterized family of RNA viruses that infect humans and a broad range of domestic animal species. The human viruses are common respiratory pathogens that cause approximately 25% of the upper respiratory infections. Coronaviruses can target to the central nervous system (CNS) and may be associated with human CNS disease. Coronaviruses uniquely assemble at membranes between the endoplasmic reticulum and Golgi. The long term objective is to understand, at the molecular level, the mechanism of coronavirus assembly. In the proposed studies both in vitro and in vivo assay systems will be used to study the role and requirements of the viral proteins and RNA in assembly. The central hypothesis is that assembly of coronavirus particles is dependent on (1) selective encapsidation of the viral RNA, and (2) a specific set of interactions between the encapsidated RNA (nucleocapsid) and the viral membrane proteins. The cytoplasmic domain of M, the protein that localizes in the budding compartment, functions like a "receptor" that interacts with the nucleocapsid to initiate assembly of virions at these membranes. The specific aims of the proposal are: (1) To map and characterize the encapsidation site on the bovine coronavirus (BCV) RNA. A recently established in vitro RNA binding assay and in vivo expression of an RNA replicon that is packaged will be used to identify and characterize the requirements for encapsidation of the BCV viral RNA. (2) To map the domains (s) on the BCV nucleocapsid protein (N) that interact with the RNA. The same in vitro RNA binding assay used in specific aim l will be used to determine the requirements for specific association of N with the viral RNA. Two phosphorylated forms of N in BCV infected cells will be investigated to gain insight into the role of phosphorylation in assembly. (3) To map the domain(s) on the integral membrane protein (M) that interacts with the nucleocapsid. The hypothesis that the M cytoplasmic domain functions like a "receptor" will be tested by measuring the binding of wild-type M and mutant forms of the protein to nucleocapsids, in vivo expression of a chimeric protein and the use of a peptide to block nucleocapsid binding.